Liquids (e.g., mixtures, solutions, biological samples) are often characterized using optical techniques such as photometry, spectrophotometry, fluorometry, or spectrofluorometry. Typically, a liquid is contained in a vessel referred to as a cell or cuvette, two or more of whose sides are of optical quality and permit the passage of those wavelengths needed to characterize the liquid contained therein. Recent applications require the characterization of very small liquid sample volumes. When dealing with very small sample volumes of, for example, from 1 to 2 microliters, it is difficult to create cells or cuvettes small enough to be filled and permit the industry standard 1 cm optical path to be used.
For instance, UV-Visible Spectrophotometry may be used to characterize the chemical composition of a liquid sample (in solution or suspension phase) using the absorbed spectra of the sample. The light absorbance of a sample depends on the pathlength L of light passing through the sample, as well as on the concentration of light absorbers (e.g., biomolecules, cells, etc) in a sample solution and the wavelength (λ) of light being used to characterize the sample. The wavelengths of UV-Visible light span from 200 nm to 800 nm, while ultraviolet wavelengths range from 200 to 400 nm.
UV-Visible spectrophotometry provides a convenient analysis technique to determine the concentration, purity, and integrity of a biological sample without requiring additional sample preparation other than acquiring a sample. UV-Visible Spectrophotometry measurements depend on the light source (UV lamp), the sample and sampling technique. Most biological samples absorb electromagnetic radiation at wavelengths ranging from 200 nm to 800 nm, mostly 230, 260 and 280 nm. For a DNA or RNA sample in aqueous phase, one unit of absorbance 1 Å measured at a λ 260 nm and a pathlength of 10 mm is equal to 50/(40) ng/μl concentration.
Most biological samples are highly concentrated for downstream processing (such as microarray spotting or protein sample preparation for mass spectrometers). The absorbance of such samples can be above the saturation limit for typical spectrophotometers if the pathlength is about 10 mm. While the sample concentration range can be extended by diluting the sample, diluting sample requires additional laboratory work and can result in errors. Other approaches are needed to extend the sample concentration range that can be evaluated by the instrument.
Sampling techniques used in conventional UV-Visible Spectrophotometers include utilizing a cuvette with an optical window and fixed optical pathlength that holds a sample in a semi-closed way, direct measurement of liquid sample in a sample container (e.g., a well) along with a real-time pathlength measurement, and using a cuvetteless sample held in semi-free space between optical fibers which define a light path from a light source to a detector.
The cuvette-based sampling technique is widely used in conventional UV-Visible spectrophotometers. Generally, a sample is pipetted into a cuvette that has either a 10 mm or 2 mm path length. This technique is very limited for most biological samples since cuvettes typically used generally require a minimum 10 μl sample, which is problematic for valuable biological samples which may be present in limiting quantities, such as samples of protein or nucleic acids. A cuvette made of quartz or silica is expensive so it is typically reused after cleaning and drying. Further, adding 10 μl of sample with a pipette into a cuvette sometimes produces an air-bubble interface in the light path which can cause measurement error or void measurements. Additionally, a pathlength of 2 mm or 10 mm limits the sample concentration which may be measured to 1000 ng/μl for a DNA/RNA sample due to the limited dynamic range of absorbance of most spectrophotometers.
In one of the existing systems that enable the measurement of very small liquid sample volumes, a sample of the liquid to be examined is inserted, by means of a dispenser needle or other means, and retained between a light transmitter and a light detector. The surface tension of a microliter or submicroliter sample of liquid is used to provide sufficient means to confine the sample between two substantially parallel surfaces on anvils spaced apart a known distance; two optical fibers penetrate the parallel surfaces and provide the light for the measurement. The electromagnetic radiation emanating from the fibers is not collimated, making the determination of path length either less accurate or complicated. In order to render one of the anvils moveable, at least one of the fibers is exposed and moveable. The bending of the fiber can introduce variable optical transmission.
Based on the above, there is a need for optical instrument designs that allow for simple and accurate optical measurements.
Regulations, such as 21 C.F.R. Part 11, have been recently released and published, enabling pharmaceutical companies to provide electronic copies of their results to regulatory agencies with electronic signatures and to rely on electronic audit trails. The regulations place high emphasis on the implementation of all measures to protect and secure electronic records. These regulations cover the basic requirements of validation, limiting data access, and ensuring data integrity and data traceability. In conventional UV-visible spectroscopy data systems, it has been uncommon to find systems that support compliance with these regulations.
Therefore, there is a need for optical instrument designs that allow for simple and accurate optical measurements and which enable the compliance with electronic record-keeping regulations.